TGFβ-induced changes in membrane curvature influence Ras oncoprotein membrane localization

In the course of cancer progression tumor cells undergo morphological changes that lead to increased motility and invasiveness thus promoting formation of metastases. This process called epithelial to mesenchymal transition (EMT) is triggered by transforming growth factor (TGFβ) but for gaining the full invasive potential an interplay between signaling of TGFβ and Ras GTPases is required. Ras proteins possess a lipidated domain that mediates Ras association with the plasma membrane, which is essential for Ras biological functions. Type and number of the lipid anchors are the main difference among three Ras variants—H-ras, N-ras and K-ras. The lipid anchors determine membrane partitioning of lipidated proteins into membrane areas of specific physico-chemical properties and curvature. In this study, we investigated the effect of TGFβ treatment on the subcellular localization of H-ras and K-ras. We show that TGFβ increases positive plasma membrane curvature, which is subsequently sensed by H-ras, leading to its elevated plasma membrane localization and activation. This observation suggests the existence of a novel positive feedback loop whereby the increased level of plasma membrane curvature during TGFβ induced EMT attracts more Ras molecules to the plasma membrane resulting in increased Ras activity which in turn promotes further EMT and thus ultimately enables the acquisition of full invasive potential.


TGFβ-1 induces increase in Ras protein PM localization. To follow subcellular localization of H-ras
and K-ras during TGFβ treatment we used breast cancer cell line MCF7 and transfected it with GFP tagged constitutively active GTP-bound H-ras G12V and K-ras G12V (Fig. 1a). The transfected cells were treated with TGFβ-1 for 2 days. The equal level of expression of fusion protein in samples with and without TGFβ-1 treatment was carefully checked and confirmed (Fig. S1a). The subcellular localization of fusion proteins was analyzed both qualitatively by visual inspection and quantitatively by calculating membrane-to-cytoplasm ratio (M-C ratio), when higher M-C ratio translated into higher accumulation of fusion protein on PM.
In case of H-ras a large proportion of cells showed cytoplasm localization in steady state and this proportion significantly decreased after TGFβ-1 treatment resulting in more than 91% of cells with H-ras on PM (Fig. 1b). In contrast, K-ras that showed a very similar proportion of cells with PM and cytoplasm localization in steady state as H-ras did not change this proportion even after TGFβ-1 addition. However, similar to H-ras, the total amount of K-ras bound to PM increased after TGFβ-1 addition (Fig. 1c, d, S2).
To study the mechanism of increased membrane localization of H-ras in more detail we tested its truncated version without the catalytic domain (G-domain). It has been documented that the minimal membrane anchor part of H-ras (tH) requires presence of adjacent hypervariable linker region to be laterally segregated as H-ras G12V and K-ras G12V into cholesterol-independent microdomains where the signaling occurs 17,19 . Therefore, we used CTH construct composed of both membrane anchor part and the hypervariable linker region tagged with CFP (Fig. 1a). Indeed, CTH followed the trend set by H-ras indicating that this membrane anchor part is sufficient for the protein response to TGFβ-1 treatment ( Fig. 1b-d, S2).
Our results thus show that TGFβ-1 triggers relocalization of H-ras from cytoplasm to PM and causes increased accumulation of K-ras in PM.
TGFβ-1 treatment triggers rise in positive membrane curvature. In a previous study we have proven on example of tN-ras, a minimal membrane anchor of the N-ras isoform, that Ras senses positive membrane curvature in in vitro reconstituted systems and that this membrane partitioning is essential for its enrichment in raft-like liquid ordered phases of membrane 13 . In addition, H-ras and to a lesser extend also K-ras were shown to preferentially localize to positively curved membranes in vivo 14 . Therefore we decided to investigate if changes in the partitioning of H-ras G12V, CTH and K-ras G12V during TGFβ-1 treatment are accompanied by the acquisition of positive membrane curvature. We transfected MCF7 cells with YFP tagged Nadrin N-BAR, a sensor of positive membrane curvature 20 , and followed changes in Nadrin N-BAR localization triggered by TGFβ-1 (Fig. S1b).
At steady state Nadrin N-BAR localized almost entirely in cytoplasm, whereas several puncta of Nadrin N-BAR accumulated on the PM after treatment with TGFβ-1 indicating that the cells acquired increased positive membrane curvature (Fig. 2).
Apart from sensing positive membrane curvature the N-BAR domains were documented to bind negatively charged lipids. N-BAR domain of another N-BAR containing protein amphiphysin was shown to interact equally well with two lipids-PI(4,5)P2 and phosphatidylserine (PS) 21 . To investigate the possible role of these proteinlipid interactions in observed responses of Nadrin N-BAR to TGFβ-1 we transfected MCF7 cells with GFP-tagged PH domain of PLCdelta, sensor of PI(4,5)P2 22 , and C2 domain of Lactadherin, specifically binding PS 23 (Fig. S1b). www.nature.com/scientificreports/ In both cases the sensors of negatively charged lipids showed mostly PM localization that either remained unchanged (PLCdelta PH) or decreased (Lactadherin C2) after TGFβ-1 treatment. Similarly, the total amount of proteins on PM after TGFβ-1 treatment showed no significant difference in case of PLCdelta PH, but decreased significantly in case of Lactadherin C2 (Fig. 2). The effect observed for Lactadherin C2 is likely to be caused by redistribution of PS from PM because a proteomic study performed in MDCK cells did not detect any significant drop in total PS amount after EMT induction 24 . It is also consistent with observation that PM localization of PS decreases with increasing positive membrane curvature whereas localization of PI(4,5)P2 is not significantly affected 14 . Since the response of Nadrin N-BAR to TGFβ-1 treatment does not follow the trend observed in either PLCdelta PH or Lactadherin C2, we can therefore conclude that TGFβ-1 induced changes in Nadrin N-BAR  Table S1). (d) Representative images of all tested conditions. Scale bar 10 μm. Increased level of positive membrane curvature leads to elevated H-ras PM localization. To validate our theory that the increase in accumulation of Ras proteins in PM during TGFβ-1 treatment is driven by the rise of positive membrane curvature we performed a set of further experiments for which we used a fibroblast cell line NIH 3T3 as an independent system. NIH 3T3 cells were selected because fibroblasts are rich in caveolae, small PM invaginations about 60 nm in size, and thus contain high number of areas with positive membrane curvature 25 . Indeed, both the M-C ratio and the proportion of cells with PM localization of Nadrin N-BAR were higher in NIH 3T3 cells than in MCF7 cells before or after TGFβ-1 treatment (Fig. S3). Similarly, both H-ras and CTH showed significantly higher association with PM in NIH 3T3 compared to situation in MCF 7 favoring the fact that H-ras and CTH also recognize positive membrane curvature (Fig. S3).
In contrast, recruitment of K-ras to PM decreased in NIH 3T3 compared to MCF7 cells (Fig. S3c). This result is consistent with previous observation in BHK cells where increase in positive membrane curvature also led to PM depletion of K-ras 14 . A possible explanation for this could lay in a different way how H-ras and K-ras associate with PM. PM localization of H-ras occurs through two lipid anchors-farnesyl and palmitoyl, whereas PM localization of K-ras, apart from its one lipid anchor (C-terminal farnesyl), largely depends on binding negatively charged lipids, especially phosphatidylserine (PS), via its polybasic domain (PBD) 26,27 . Similar to K-ras, Lactadherin C2 showed also significantly reduced PM binding (Fig. S3e). Moreover, it was recently demonstrated that K-ras G12V strictly prefers PS with unsaturated acyl chains over fully saturated PS 28 . Since caveolae are known to be composed mostly of lipids with saturated acyl chains, pool of unsaturated PS available for binding may be  Comparison of membrane-to-cytoplasm ratios (M-C ratio) (Nadrin N-BAR n ≥ 132, PLCδ PH n ≥ 96, Lactadherin C2 n ≥ 188), error bars represent ± 0.5 SD, stars indicate statistically significant differences (*p = 0.01-0.05, **p = 0.001-0.009, ***p < 0.001; for exact p-values see Table S2). (c) Representative images of all tested conditions. Scale bar 10 μm. www.nature.com/scientificreports/ actually significantly smaller in caveolae-rich fibroblasts compared to epithelia cells and, in combination with overall lower PS level, it could represent a main limiting factor for K-ras G12V PM recruitment in NIH 3T3 cells.
To inspect if Ras proteins indeed preferentially localize to areas of high positive membrane curvature we co-transfected H-ras G12V with Nadrin N-BAR in NIH 3T3 cells and we observed a significant correlation in PM localization of both proteins (Fig. 3).

Disruption of membrane curvature causes drop in Ras proteins PM localization. The results of
our experiments strongly suggests that recognition of positive membrane curvature is likely a driving mechanism behind increased PM targeting of Ras after TGFβ-1 treatment. Therefore, experiments reducing positive membrane curvature of cell membranes should cause release of Ras from PM.
One option how to reduce membrane curvature is to subject cells to hyposmotic shock. The cells start to swell as indicated by increased FM1-43 staining (Figs. 4a, b, S4) 29 . Moreover, osmotic swelling leads to rapid disappearance of caveolae which is more prominent the lower the osmolarity gets. For our experiments we thus used hyposmotic level shown to be required to reduce caveolae by ~ 30% 30 . Drop in Nadrin N-BAR PM localization indeed confirmed that hyposmotic shock reduces positive membrane curvature in NIH 3T3 cells. H-ras and CTH were both rapidly released from PM following hyposmotic shock induction with response of CTH being more pronounced than the one of H-ras. K-ras also showed fast and stable release from plasma membrane (Figs. 4b,  S4). Interestingly, the hyposmotic shock decreased the level of Lactadherin C2 bound to PM as well which is in contradiction with observation that PM localization of PS decreases with increasing positive membrane curvature (Fig. 2, and 14 ). One possible explanation could be that PS in PM respond differently to changing membrane curvature under different conditions. Alternative explanation could be that, in addition to stretching the membrane and disappearance of caveolae, the high hyposmotic stress may disrupt the global organization of the membrane bilayer which may then contribute to the loss of interactions of some proteins with PM 14 . Both of these possibilities could provide an explanation to the release of Lactadherin C2 from PM following hyposmotic shock and may contribute also to the release of K-ras taking into account the dependence on PS binding of both of these proteins (Figs. 4b, S4). The drop in K-ras PM localization was much higher than in case of Lactadherin C2 suggesting that reduction in PM PS level is likely not the only reason for K-ras release from PM and that reduction in positive membrane curvature also plays its role. However, the different level of response of K-ras and Lactadherin C2 to the hyposmotic shock may have one more explanation. It was previously shown that distinct PS species prefer membranes of different curvature depending on their acyl chains-fully saturated and mono-unsaturated PS species favor highly curved membranes, while the mixed-chain PS species prefer less curved membranes 14 . The distinct PS pools in PM may respond to changes in membrane curvature in opposing ways, yielding a more subtle www.nature.com/scientificreports/ response of the global PS localization on the PM (sensed by Lactadherin C2) in comparison with K-ras G12V which was shown to prefer PS with unsaturated acyl chains over fully saturated PS 28 . In contrast to Nadrin N-BAR that showed constant and gradual decrease in PM localization, the release of Ras isoforms from plasma membrane reaches sooner or later plateau reflecting possibly different ways of interaction with PM and membrane curvature recognition by these proteins (concave shape of N-BAR vs. lipid anchors of Ras) (Fig. S4).
Besides the hyposmotic shock that disturbs membrane curvature in general, positive membrane curvature can be specifically reduced by targeted lowering of the caveolae number. Expression of dominant negative caveolin (Cav DGV ) was shown to significantly reduce number of caveolae (reduction by 62% in BHK cells) 31 . We performed co-expression of GFP-Cav DGV with RFP-H-ras G12V in NIH 3T3 cells followed by quantitative analysis of H-ras PM localization. Indeed, we observed a decrease in H-ras PM localization when single (RFP-H-ras G12V) and double (GFP-Cav DGV with RFP-H-ras G12V) transfected cells were compared (Figs. 5, S5). The reduction was partial because, consistently with previous observations, H-ras was detectable on PM even at high expression levels of Cav DGV (Figs. 5, S5); 31 . However, the effect of Cav DGV expression on the PM localization of H-ras was even more evident from the negative correlation between the amount of H-ras associated with PM and the level of Cav DGV expression (Figs. 5, S5).

Discussion
Ras proteins are small GTPases that serve as important regulators of cell pathways responsible for proliferation, differentiation and cell survival. Mutations in key conserved sites within Ras proteins lead to elevated GTP binding which results in constitutive activation of Ras. Among the three Ras isoforms, mutations in K-ras are the most frequently detected in human cancer, but specific associations of individual mutated Ras isoforms with particular cancer types were detected 3,4 .
Similarly as Ras proteins, TGFβ-1 is frequently overexpressed in human tumours 32 and its expression is generally associated with poor prognosis 33,34 . One of the functions of TGFβ-1 is to induce EMT, which is important biological process critical during embryogenesis, but it is also exploited by cancer cells during tumor progression. TGFβ-1 signal alone was shown to be insufficient for acquisition of invasive potential of cancerous cells. To gain full invasive potential activated Ras (H-ras) that alters TGFβ-1 response is needed 10 . Moreover, activated Ras (H-ras) or its downstream effectors are important for promoting EMT through autocrine production of TGFβ-1 and continuous TGFβ-1 signaling 8,9 . For proper Ras signaling its PM localization is essential. In this study we showed that TGFβ-1 treatment of breast cancer cells MCF7 is followed by elevated PM localization of activated H-ras and K-ras. Suggesting that TGFβ-1 itself can promote Ras PM residence. The driving mechanism behind this seemed to be triggered by TGFβ-1 and manifested by alterations in membrane curvature resulting in increased level of positive membrane curvature. The recognition of membrane curvature by activated Ras is likely important for proper signaling, because it is known that Ras signaling activity is localized mostly at the periphery of a cell or the leading edge of a migrating cell, where membrane ruffling is prominent in both cases 35,36 .
Our results provide evidences supporting hypothesis that Ras proteins are indeed able to react to changes in membrane curvature in vivo which is in agreement with recent observations by others 14 . The membrane anchor part of H-ras seems to be central and sufficient to H-ras recognition of positive membrane curvature. H-ras, containing two lipid anchors, seems to be also more potent sensor of positive membrane curvature than K-ras that contains only one lipid anchor, which is consistent with our previous finding that the presence of more lipid anchors leads to higher sensitivity to positive membrane curvature 12 . Active K-ras indeed prefers less curved membranes than active H-ras even in vivo and increase in positive curvature rather leads to its disappearance from PM 14 . Despite the fact that PM localization of K-ras largely depends on the presence of PS, its membrane  www.nature.com/scientificreports/ partitioning driven by TGFβ-1 treatment seems to be at least partially PS independent as the amount of K-ras on PM increases during TGFβ-1 treatment, whereas amount of specific PS sensor Lactadherin C2 decreases ( Figs. 1 and 2, S2). The effect of TGFβ-1 treatment on Ras PM localization described in this paper suggests a simple mechanism of possible positive feedback loop within previously described TGFβ-1 and Ras cooperation during cancer progression. In this hypothesis an increased level of membrane curvature of PM during TGFβ-1 induced EMT would attract more Ras molecules to PM resulting in its facilitated activation and subsequent further promotion of EMT and acquisition of invasive potential (Fig. 6). However, further validation of this theory would be needed. Especially a detailed insight into the molecular mechanism behind observed phenotypes would bring broader understanding of the suggested link between membrane curvature sensing of Ras proteins and TGFβ-1 induced EMT and could be achieved for example via studying the process after inhibition of TGFβ-1 induced signaling pathways 37,38 . It is noteworthy that our experiments with CTH suggest that the hyperactivating G12V mutation is not necessary for curvature coupling of H-Ras. The novel TGFβ-EMT feedback loop we propose here, which leads to H-Ras membrane localization and activation, is mediated by the membrane curvature biophysical properties of the CTH anchor. The mechanism of membrane-curvature-based H-Ras activation is thus, in principle, not predicated on the existence of mutations. However, this mechanism will be particularly sensitive to stimuli that modify plasma membrane morphology during both development and tumor formation 39,40 . Last but not least, it is important to highlight that the pathological relevance of the observed effect of TGFβ-1

Plating of cells and transfection.
Cells were grown in 6 well plates on round cover slips (Ø 18 mm, VWR) that were thoroughly cleaned using 2% Hellmanex III (Hellma ® Analytics) and MetOH prior use. In case of MCF7 cells 6 × 10 4 of cells were plated per well, in case of NIH 3T3 5 × 10 4 of cells were plated per well. For co-transfection experiment of GFP-Cav DGV with RFP-H-ras G12V 10 × 10 4 NIH 3T3 cells were plated per well. Cells were grown for 24 h in culturing medium before transfection. For co-transfection experiment of RFP-Hras G12V and Nadrin-YFP N-BAR plasmids 5 × 10 4 of cells were plated and 1 μg of each plasmid was used. The transfection was performed using Lipofectamine plus (Invitrogin) according to manufacturer's instructions.
TGFβ-1 treatment. 24 h after transfection the transfected cells were placed in fresh culturing medium supplemented with 2 ng/ml of TGFβ-1(R&D systems) and incubated in this medium for two days.
Live cell imaging. For imaging the cover slips were mounted in custom made microscopy chambers with total volume of 90 μL. Cells were imaged in imaging medium (DMEM without phenol red with 10% FBS) using Leica TCS SP5 inverted confocal microscope with a water immersion objective HCX PL APO CS × 63 (NA 1.2). Signal from GFP was detected at 495-590 nm (exc. 488 nm) and YFP was detected at 520-538 nm (exc. 514 nm); CFP was detected at 468-590 nm (exc. 458 nm); RFP was detected at 565-699 nm (exc. 543 nm); FM 143 was detected at 560-610 nm (exc. 471 nm). In case of co-expression of proteins with two different fluorescent tags sequential imaging was used to avoid cross excitation. Images had a resolution of 2048 × 2048 pixels, with a pixel size of 120 nm and a 16-bit depth. All acquired images were then processed with open source software Fiji.
Hyposmotic shock. We imposed hyposmotic shock following the protocol described in 20 . Cover slips with transfected NIH 3T3 cells were mounted into microscopy chambers and imaged (sequential imaging, one image taken every 30 s). After one minute of imaging the full chamber volume (90 μL) of imaging medium was replaced with imaging medium diluted with milliQ water in ratio 1:6 (from 300 to 50 mM DMEM) and the sample was imaged further for another 5 min. The control sample was treated in the same way except that the imaging medium was replaced with 90 uL of fresh non-diluted imaging medium. Membrane swelling after application of diluted medium was checked visually and also by plasma membrane staining with FM 143 dye (Thermo Fisher Scientific). The staining was performed according to manufacturer's instructions.
Quantitative image analysis. Quantitative image analysis procedures were performed in IgorPro (version 6.37, Wavemetrics, USA). Only healthy cells in focus and with clear borders (identified by eye) were used for the analysis. First to visualize cell membrane better and to reduce noise images were convoluted with a Gaussian blur filter (5 pixels size). Then line ROI (5 pixels width) was drawn manually on top of the plasma membrane. This line ROI allowed us to isolate pixels in image belonging to the plasma membrane and cytoplasm. The line ROIs were always drawn as close to cell interior as possible. In this way we minimized the potential impact of www.nature.com/scientificreports/ the fluorescent signal coming from the adjacent cell(s) on plasma membrane intensity value of the cell of interest in cases when two or more cells were in close contact. Finally, based on the line ROI, statistics of fluorescence intensity in the plasma membrane and cytoplasm pixels were calculated using the raw image. The membrane and cytoplasm intensity ratio (M-C ratio) was assessed by dividing average membrane intensity by average cytoplasm intensity. To find whether the difference of M-C ratio between two samples is statistically significant, we performed two-tailed T-test (significance level = 0.05, equivalent degrees of freedom accounting for possibly different variances). For analysis of data from hypoosmotic shock experiment integrated intensity of the membrane was used for making intensity versus time traces. In order to be able to compare the osmotic shock effect between different samples each trace was normalized to the average intensity of the first three points from the beginning of the trace. Next an average intensity for first 3 points (I0) and from point 4 till the end of the trace (I1) was calculated and used for the percentage intensity change ( I ) calculation. Then I = I1−I0 I0 * 100 was calculated for each individual cell and their average and SD was taken.
To analyse co-expression experiment of GFP-Cav DGV with RFP-H-ras G12V in NIH 3T3 cells the linear correlation test was performed and the linear correlation coefficient and its standard error were calulated to estimate the degree of correlation between H-ras M-C ratio and the total Cav DGV expression. The significance level was set to 0.05, that gave confidence intervals for the correlation coefficient at 95%.
Qualitative image analysis. Qualitative image analysis was based on visual inspection of acquired images.
According to localization of fluorescent signal coming from expressed fusion proteins the cells were manually classified as showing cytoplasm localization (i.e. fluorescent signal was visible only in cytoplasm with no plasma membrane localization) or plasma membrane localization (i.e. fluorescent signal was localized entirely or at least partially on plasma membrane) of expressed protein. Only healthy cells in focus and with clear borders were assessed in the analysis.